Cellular Environment Modestly Affects Binding Dynamics

Protein-binding rate constants have typically been measured in vitro under dilute conditions. But scientists have begun to question whether such conditions accurately reflect the crowded environment inside living cells. A study suggests that they may not need to worry (Proc. Natl. Acad. Sci. USA, DOI: 10.1073/pnas.1112171109). Gideon Schreiber, Yael Phillip, and Vladimir Kiss of Weizmann Institute of Science, in Rehovot, Israel, used distance-sensitive fluorescence resonance energy transfer (FRET) imaging to measure binding dynamics between two proteins, β-lactamase and its inhibitor BLIP, expressed as fusions with fluorescent proteins. Because FRET is sensitive to the distance between fluorophores, the fluorescence can be used to measure how quickly β-lactamase and BLIP bind. The association rate constants were only 25% and 50% lower for the wild-type proteins and an optimized mutant inhibitor, respectively, compared with the in vitro values. In this system, “the intracellular environment had only a small effect on the association kinetics, justifying the extrapolation of in vitro data to processes in the cell,” the researchers write.